Experiment | Substrate | Feeding strategy based on qs [g g−1 h−1]a | Goals |
---|---|---|---|
FB1 | Glycerol | Batch on glycerol Fed-batch on glycerol with step-wise decrease of qs,gly 0.567–0.450–0.282–0.142–0.101–0.070–0.050 g g−1 h−1 (each step was done for 2 h) Methanol pulses: 0.5% (v/v) adaptation pulse at 30 °C—two 2% (v/v) pulses at 30 and 20 °C, respectively | Determination of strain specific physiological parameters (qs,max,gly; qs,gly for maximum qp; qs,adapt,MeOH; qs,max,MeOH) Characterization of recombinant expression profile at different process parameters |
FB2 | Glycerol and methanol | Batch on glycerol 0.5% (v/v) methanol adaptation pulse Constant MeOH feed at qs 0.02–0.025 g g−1 h−1 and concomitant glycerol feed with stepwise increases: 0.025-0.059-0.139-0.239 g g−1 h−1 (each step was done for 2 h) | Find operating window for maximum qp |
FB3 | Glycerol and methanol | Batch on glycerol Fed-batch at qs,gly = 0.212 g g−1 h−1 0.5% (v/v) methanol adaptation pulse followed by a fed-batch on methanol at qs = 0.034 g g−1 h−1 Mixed feed cultivation at qs,gly = 0.135 g g−1 h−1 and qs,MeOH = 0.035 g g−1 h−1 | Demonstrate tunability of recombinant protein production |